REVIEW

VEGF-A and blood vessels: a beta cell perspective

Willem Staels1,2 & Yves Heremans1 & Harry Heimberg1& Nico De Leu1,3,4

Received: 18 March 2019 /Accepted: 11 June 2019# Springer-Verlag GmbH Germany, part of Springer Nature 2019

AbstractReciprocal signalling between the endothelium and the pancreatic epithelium is crucial for coordinated differentiation of theembryonic endocrine and exocrine pancreas. In the adult pancreas, islets depend on their dense capillary network to adequatelyrespond to changes in plasma glucose levels. Vascular changes contribute to the onset and progression of both type 1 and type 2diabetes. Impaired revascularisation of islets transplanted in individuals with type 1 diabetes is linked to islet graft failure andgraft loss. This review summarises our understanding of the role of vascular endothelial growth factor-A (VEGF-A) andendothelial cells in beta cell development, physiology and disease. In addition, the therapeutic potential of modulating VEGF-A levels in beta and beta-like cells for transplantation is discussed.

Keywords Blood vessels . Diabetes . Endothelial cells . Pancreatic beta cells . Review . VEGF-A

AbbreviationsAng AngiopoietinBOEC Blood outgrowth endothelial cellE Embryonic dayFGF Fibroblast growth factorHGF Hepatocyte growth factorIBMIR Instant blood-mediated inflammatory responseRTK Receptor tyrosine kinaseVEGF Vascular endothelial growth factorVEGFR Vascular endothelial growth factor receptor

VEGF-A and the VEGF family

Vascular endothelial growth factors (VEGFs) are broadlyexpressed dimeric molecules that are structurally related byeight conserved cysteine residues that form a typicalcysteine-knot structure [1]. VEGF-A is the founding memberof the VEGF family, which also includes placental growthfactor (PlGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E andsnake venom (sv)VEGF [1]. All VEGF family members sig-nal by binding to cell surface receptor tyrosine kinases (RTKs)[2]. VEGF-A binds several RTKs, including VEGF receptor-1(VEGFR-1, also known as Flt-1) and VEGFR-2 (also knownas KDR or Flk-1), both expressed predominantly on endothe-lial cells that constitute the lining of blood vessels [2].Although VEGF-A has higher affinity for VEGFR-1, its mainsignalling effects are conferred through VEGFR-2 [2]. In de-velopment and physiology, VEGF signalling coordinatesvasculogenesis and angiogenesis by guiding endothelial cellproliferation, migration and survival, as well as vascular per-meability [2]. Not surprisingly, aberrant VEGF signalling istherefore implicated in the pathophysiology of several dis-eases, including diabetes [2].

VEGF-A and blood vessels in pancreasdevelopment

Reciprocal interactions between endothelial cells and othercell types occur during ontogeny, specification and branching

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s00125-019-4969-z) contains a slide of thefigure for download, which is available to authorised users.

* Nico De LeuNico.De.Leu@vub.be

1 Beta Cell Neogenesis (BENE), Vrije Universiteit Brussel,Laarbeeklaan 103, 1090 Brussels, Belgium

2 Institut Cochin, CNRS, INSERM, Université de Paris,F-75014 Paris, France

3 Department of Endocrinology, UZ Brussel, Brussels, Belgium4 Department of Endocrinology, ASZ Aalst, Aalst, Belgium

https://doi.org/10.1007/s00125-019-4969-zDiabetologia (2019) 62:1961–1968

/Published online: 14 August 2019

morphogenesis of a variety of tissues and organs, includingthe liver [3] and lung [4]. Likewise, vascular-derived signalsplay a pivotal role during pancreas development (Fig. 1).Pioneering work by Lammert et al showed that non-nutritional signals derived from aortic endothelial cells induceendocrine pancreas differentiation [5]. The dorsal endodermof embryonic mice devoid of VEGFR-2 lacks endothelial cellsand does not express Ptf1a, Ins1 or Gcg [6]. Vascular signalstake part in a relay pathway from the aortic endothelium to thedorsal pancreatic mesenchyme and, through fibroblast growthfactor (FGF)-10 signalling, from the mesenchyme to the en-doderm. These interactions are crucial for the maintenance ofPtf1a expression and subsequent endoderm differentiation inthe dorsal pancreas of embryonic day 9.5 (E9.5) [7].

While needed for induction of embryonic pancreas spec-ification, blood vessels restrain outgrowth and morphogen-esis of the pancreas epithelium during later development.

The trunk region (where bipotent progenitors of endothelialand duct cells reside) expresses higher levels of VEGF-A ascompared with the tip epithelium (where acinar progenitorsreside), resulting in increased vascularisation and decreasedbranching as compared with the tip epithelium [8]. VEGF-A loss- and gain-of-function experiments in E12.5 pancreasrevealed that hypovascularisation favours pancreaticbranching and both exocrine and endocrine cell differenti-ation through the induction of carboxypeptidase A1(CPA1)+ and PTF1a+ multipotent tip cells and ofneurogenin 3 (NGN3)+ endocrine progenitor cells, respec-tively, while hypervascularisation sustains the primitive un-differentiated state of the tubular trunk epithelium [9].Overexpression of VEGF-A in developing beta cells resultsin reduced beta cell proliferation at E16.5 and postnatal day1 (P1) and in impaired islet cell clustering, culminating in areduced postnatal beta cell mass. In contrast, increased

a

b

c

Fig. 1 The impact of loss or gain of blood vessels on pancreas develop-ment and function. (a) Loss of blood vessels, (b) normal vascularisation,and (c) gain of blood vessels. Blood vessels provide non-nutritional cuescrucial for endocrine pancreas development and function. Loss of bloodvessels during early pancreas development (~E9.5) inhibits normal pan-creatic bud outgrowth and endocrine cell formation. Later on (~E12.5),hypovascularisation promotes pancreatic branching and exocrine and en-docrine cell differentiation, while hypervascularisation sustains the

undifferentiated state of the tubular trunk epithelium. In the adult pancre-as, islet hypovascularisation causes islet hypoxia and mild glucose intol-erance, but the direct, deleterious effects on the beta cell are small. Adultislet hypervascularisation has time-dependent effects on the beta cell,initially increasing beta cell proliferation and function, while later onpromoting islet inflammation and loss of beta cells. This figure is avail-able as a downloadable slide

Diabetologia (2019) 62:1961–19681962

expression of angiopoietin (Ang)1 or Ang2 in developingbeta cells does not alter islet cell differentiation or morphol-ogy [10].

VEGF-A and blood vessels in adult beta cellphysiology

While islets of Langerhans in the adult pancreas are highlyvascularised in general [11], species-specific differences exist[12, 13]. Compared with humans, in the mouse islet vasculardensity is substantially higher and more tortuous [12, 13] andintra-islet blood vessels contain fewer smooth muscle cells[11]. Furthermore, mouse islet endocrine cells lack a basementmembrane and directly interact with the vascular endothelialbasement membrane, while in humans the islet endocrine cellsare separated from the endothelial cells by a double basementmembrane [14]. Several mechanisms, including angiopoietin–Tie2 signalling [10] and intra-islet biomechanical properties[15], are involved in the regulation of islet vessel density andin the crosstalk between islet cells and endothelial cells, butthe role of VEGF-A has been studied most extensively. Bothin human and mouse endocrine pancreas, VEGF-A is mainlyproduced by alpha and beta cells, functioning as an importantregulator of the islet microvasculature [16]. Vegfa knockout ininsulin-producing cells (RIP-Cre;VegfloxP/loxP mice) results inislet hypovascularisation, loss of endothelial fenestrations anddecreased glucose clearance and glucose-stimulated insulinsecretion but does not affect adult beta cell mass [16, 17] norits capacity for expansion in response to insulin resistance,when stressed by a high-fat diet [18]. In line with these find-ings, we showed that adult mice with reduced intra-isletVEGF-A bioavailability through soluble (s)VEGFR-1-medi-ated scavenging show a rapid regression of intra-islet bloodvessels with ensuing islet hypoxia, together with slightly in-creased basal and stimulated blood glucose levels [19]. Thebeta cell mass and basal beta cell proliferation of the miceremain unaffected. Furthermore, intra-islet hypovascularisationdid not prevent beta cell proliferation after severe pancreas in-jury caused by partial pancreatic duct ligation or partial pancre-atectomy [19].

The islet vasculature in adult mice undergoes dynamic andmorphological changes in response to fluctuations in bloodglucose levels and to intra-islet VEGF-A and insulin levels.Low glucose reduces intra-islet blood flow and decreasesVEGF-A secretion in both cultured islets and purified betacells [20]. Sustained hypoglycaemia results in apoptosis of

endothelial cells and beta cells and in a reduction in beta cellmass which could be rescued by exogenous delivery ofVEGF-A [21]. In contrast, hyperglycaemia by glucose clampor by i.v. glucose injections significantly increases intra-isletblood flow [22]. Insulin plays a crucial role in these glucose-mediated vascular adaptations since endothelial cell-specificinsulin receptor knockout mice display decreased islet bloodflow, blunted glucose-stimulated insulin secretion and glucoseintolerance [23]. Mechanistically, vascular endothelial cell-specific phosphoinositide 3-kinase (PI3K) downstream sig-nalling supports a normal islet vascular density and islet bloodflow, in addition to maintaining a normal beta cell mass andfunction [24]. Of note, several other factors including gastro-intestinal hormones, neurotransmitters and locally acting au-tacoids can influence islet blood flow [11].

VEGF-A and blood vessels in beta celldysfunction and diabetes

Type 2 diabetes is characterised by insulin resistance and betacell dysfunction [25]. Vascular changes in peripheral insulin-sensitive tissues, including decreased capillary density andrecruitment, impaired blood vessel relaxation and proinflam-matory activation of the endothelium, have been implicated inthe pathogenesis of insulin resistance [26]. Although insulinresistance significantly affects the beta cell phenotype and canultimately lead to beta cell dysfunction and exhaustion [25],elaborating on the peripheral vascular changes associated withinsulin resistance falls beyond the scope of this review.

In addition, within the islets, a link between islet vesseldensity and beta cell dysfunction has been described. Islethypovascularisation was correlated with beta cell loss anddiabetes in Zucker diabetic fatty rats [27] and Otsuka Long-Evans Tokushima Fatty rats [28], while mice fed a high-fatdiet over 4 months showed increased islet VEGF-A contentand islet hypervascularisation [29]. These findings were fur-ther elaborated by transgenic and viral vector-mediated over-expression of VEGF-A in beta cells, which resulted in isletendothelial cell accumulation, vascular leakage, inflammationand a subsequent reduction of the beta cell mass with onset ofdiabetes [29]. Consistent with these findings, we and otherinvestigators observed a reduction in beta cell mass with en-suing glucose intolerance upon extended VEGF-A overex-pression in beta cells [30, 31]. These findings in rodents arein line with observations in the pancreas of individuals withtype 2 diabetes showing increased intra-islet capillary density,

VEGF-A signalling and endothelial cells are crucial for

pancreas specification, branching morphogenesis

and both endocrine and exocrine pancreas differenti-

ation.

VEGF-A and insulin signalling are crucial for adult is-

let vessel maintenance, blood flow and tight glucose

control.

Diabetologia (2019) 62:1961–1968 1963

capillary thickening and fragmentation and islet fibrosis [13,32]. Consequently, both islet hypo- and hypervascularisationseem to be correlated with beta cell mass loss in rodent modelsof type 2 diabetes. Besides changes in islet vessel density,alteration in expression of genes regulating islet blood flowand inflammation have been described in intra-islet endothe-lial cells in rodent models of type 2 diabetes [33, 34](summarised in Table 1). In addition, chronic hyperglycaemiaresults in the accumulation of AGEs that contribute to endo-thelial cell dysfunction in both db/db mice [33] and in type 2diabetic patients [35]. Mechanistically, AGEs stimulateNF-κB activation, subsequent cyclooxygenase (COX)-2 acti-vation and increased prostaglandin E2 synthesis resulting inendothelial cell dysfunction and apoptosis [36].

Type 1 diabetesmellitus is characterised by an autoimmunedestruction of insulin-producing beta cells [37]. In the NODmouse, a murine model of type 1 diabetes, this autoimmuneprocess starts with a non-destructive stage characterised by anintact peri-islet membrane surrounded by predominantlyantigen-presenting macrophages and dendritic cells, and lateron by CD4 T cells, CD8 T cells and B cells [38]. This stageprogresses to a second destructive phase during whichcathepsin-producing macrophages destroy the peri-islet mem-brane. Next, autoreactive T cells are activated by antigen-presenting cells in pancreatic lymph nodes and migrate to-wards the islets to induce beta cell destruction, ultimatelyleading to clinical diabetes [39, 40]. Aberrant islet microvas-culature has been reported at this stage [41]. The endothelialcell layer that normally forms a barrier to blood leucocytesnow displays antigen-presenting capacities and becomes per-meable, thereby allowing T cells to extravasate into the isletsand destroy them [41]. Chemokine-expressing perivascularCD11+ cells contribute to this process of T cell extravasationinto islets [42]. Interestingly, increased VEGF-A expressionoriginating from beta cells and inflammatory cells, includingCD11c+ cells, promotes islet vascular remodelling in NODmice. It has been reported that experimental interference withendothelial VEGFR-2 signalling prevents the increase in isletvascularity seen in untreated NOD mice, and impairs T celltrafficking, reverses insulitis and restores normal glucose con-trol [43]. In contrast, other investigators found a decrease inislet vessel density in NOD mice [44]. Interestingly, immuneintervention with anti-CD3 antibodies can normalise glucosecontrol in NOD mice but fails to correct islet vessel density[44]. Both islet vascularisation and glucose tolerance wererestored by increasing intra-islet VEGF-A levels through dailyglucose injections for 14 days [44]. Finally, although few re-ports exist on the intra-islet vascular adaptations in the pan-creas of patients with type 1 diabetes, islet vessels were re-cently shown to be smaller and more numerous than thoseobserved in non-diabetic control individuals [45].

Blood vessels in beta cell (re)generation

We showed that short-term overexpression of VEGF-A inadult mouse beta cells indirectly stimulates their proliferationand protects them against alloxan-mediated diabetes [31].Prolonged VEGF-A overexpression adversely results in mu-rine beta cell loss and impaired glucose clearance [30].However, restoring intra-islet VEGF-A levels to normal

Table 1 Upregulated genes in islet endothelial cells of rodent type 2diabetes models

Process Genes

Inhibition of fibrinolysis Pai-1

Vascular adhesion Sele

Pecam-1

Vcam-1

Renin–angiotensin system Ace-1

Agtr-1a

Vascular tone/oxidative stress/angiogenesis Cox-2

Nos3a

Edn1

Ho-1

Hif-1a

Nox-2

Ptgis

Cytokines and growth factors Il1b

Il1ra

Il6

Tgf-b

Tnf-a

Chemokines Cxcl1

Ccl2

Mip-1a

Cellular pathways for cytokines and TLRs Casp1

Tlr2

Tlr4

Myd88

NF-κB

iNOS

Ho-1 is also known as Hmox1; iNOS is also known as Nos2; Mip-1a isalso known as Ccl3; NF-κB is also known as Rela; Nox-2 is also knownas Cybb; Pai-1 is also known as Serpine1

Compilation of the results reported byHogan et al. [33] and Lacraz et al. [34]a Increased in [34] but not different in [33]

TLR, Toll-like receptor

Vascular changes contribute to the onset and pro-

gression of type 1 and type 2 diabetes, and VEGF-A

functions within a narrow physiological range to main-

tain islet homeostasis and function.

Diabetologia (2019) 62:1961–19681964

restores beta cell mass, function and islet structure. The re-maining beta cells increase their proliferation rate and theoverload of islet endothelial cells disappears [30]. This regen-erative process depends on the recruitment of bone marrow-derived macrophages and on endothelium-derived factors, in-cluding FGF-1, insulin-like growth factor (IGF)-1 andplatelet-derived growth factor subunit B (PDGFB) [30].

Several other endothelial-derived factors have been suggestedto modulate beta cell proliferation and function. In the endocrinepancreas of mice, endothelial cells establish a vascular niche byforming a basement membrane for endocrine cells. Herein, en-dothelial laminins connect with β1-integrins on the beta cellsurface to promote insulin gene expression and beta cell prolif-eration [46]. Also, vascular-derived collagen IV interacts withβ1-integrins on the beta cells to promote endocrine cell motilityand function [47]. Aside from such direct crosstalk, paracrineinteractions between endothelial cells and beta cells have beendescribed. Endothelial cells produce connective tissue growthfactor (CTGF) that, in turn, can upregulate positive cell cycleregulators and other factors involved in beta cell proliferation,including β1-integrin and hepatocyte growth factor (HGF) [48,49]. Of note, VEGF-A and insulin can trigger the release of HGFfrom endothelial cells which, in a co-culture system, stimulatesrat beta cell proliferation [50]. The authors suggested that thisVEGF-A/insulin–HGFpathway contributes to compensatory be-ta cell expansion during pregnancy [50]. However, in our modelof conditional islet hypovascularisation through blockingVEGF-A signalling, pregnancy-induced beta cell proliferation and ex-pansion was not precluded [51]. As we did not ablate all intra-islet endothelial cells; however, we cannot unequivocally excludethat the residual islet endothelial cells suffice for and contribute topregnancy-induced beta cell adaptation.

Blood vessels in beta cell transplantation

Transplantation of islet(-like) cells is a promising therapy forpatients with type 1 diabetes [52]. Despite a significant improve-ment in metabolic outcome compared with the originalEdmonton protocol, only 40–50% of patients remain insulin-independent 5 years after primary islet cell transplantation [53].Although the mechanism of graft failure remains largely un-known, more than half of the grafted islets are destroyed duringthe first few days after transplantation [54]. Factors implicated inearly graft loss include glucotoxicity, lipotoxicity, immune rejec-tion, toxicity of the immune-suppressive regimens, instant blood-mediated inflammatory response (IBMIR), liver ischaemia and

insufficient graft revascularisation [55]. Revascularisation oftransplanted islets takes several weeks and remains suboptimalcompared with that of endogenous islets [12]. Endothelial cellspresent in freshly isolated donor islets contribute to graftrevascularisation though inosculation with the host vasculature.When donor mouse islets are cultured before transplantation,their revascularisation solely depends on the ingrowth of recipi-ent microvessels [56].

Animal studies have demonstrated that strategies that promoteearly graft revascularisation and counteract liver ischaemia aftertransplantation can improve the outcome of islet transplantation.Overexpression of VEGF-A in grafted islets resulted in increasedvascularisation and islet blood flow, increased beta cell survivaland islet insulin content and better glucose control in recipientanimals [57]. Similar observations were made in mice withstreptozotocin-induced diabetes when the angiogenic factorAng-1 was overexpressed in transplanted islets by adenovirus-mediated gene delivery [58]. Interference with the angiostaticfactor thrombospondin-1 in transplanted islets improved graftvascularisation and function, further supporting the idea that pro-moting early graft vascularisation in clinical islet transplantationcould positively influence its outcome [59]. As genetic modula-tion of angiogenic or angiostatic factors in transplanted islets isprecluded for clinical use, alternative strategies to improve isletrevascularisation are needed. Indeed, vascular engraftment re-lates to transplantation outcome, as shown followingpreincubation of mouse or human islets with the pro-angiogenic factor prolactin before transplantation and by injec-tion of prolactin during the first 7 days after islet transplantationbeneath the renal capsule in mice [60]. Also, when pro-angiogenic cells were co-engrafted with islets, isletrevascularisation was promoted. Rat aortic endothelial cells sup-ported islet isograft survival [61], while human cord blood-derived endothelial progenitor cells improved porcine islet grafttransplantation into diabetic nude mice by accelerating graftrevascularisation [62]. Besides supporting graft revascularisation,endothelial (progenitor) cells have also been implicated in sup-pressing the detrimental effects of IBMIR [63]. Blood outgrowthendothelial cells (BOECs) are a late-outgrowing subtype of en-dothelial progenitor cells with beneficial effects on woundrevascularisation and healing [64]. We showed that humanBOECs improve islet graft function when co-transplanted withrat islets in immune-deficient diabetic mice [65]. BOEC recipi-ents had a better metabolic outcome, less beta cell death, moreproliferating beta cells in the graft, and higher graft-vessel andbeta cell volumes. This advantage was maintained with BOECsderived from a patient with type 1 diabetes [65]. In an alternativeapproach, we circumvented the limited clinical translational po-tential of viral vector-mediated gene delivery, as discussed above,by liposome-mediated delivery of Vegfa mRNA to peripheralislet cells prior to transplantation. Vegfa mRNA transfection sig-nificantly improved both mouse and human islet graftrevascularisation [66]. Such an approach using in vitro

Beta cell proliferation can be supported by endothe-

lium-derived factors and by VEGF-A, the latter in a

dose- and time-dependent manner.

Diabetologia (2019) 62:1961–1968 1965

revascularisation and healing [64]. We showed that humanBOECs improve islet graft function when co-transplanted withrat islets in immune-deficient diabetic mice [65]. BOEC recipi-ents had a better metabolic outcome, less beta cell death, moreproliferating beta cells in the graft, and higher graft-vessel andbeta cell volumes. This advantage was maintained with BOECsderived from a patient with type 1 diabetes [65]. In an alternativeapproach, we circumvented the limited clinical translational po-tential of viral vector-mediated gene delivery, as discussed above,by liposome-mediated delivery of Vegfa mRNA to peripheralislet cells prior to transplantation. Vegfa mRNA transfection sig-

nificantly improved both mouse and human islet graftrevascularisation [66]. Such an approach using in vitro tran-scribed mRNA for gene delivery has important advantages overclassic gene therapy: (1) the overexpression is transient thusavoiding side effects of chronic VEGF-A overexpression suchas increased vascular permeability and inflammation, and (2) theinherent risks of viral vector-based methods, such as insertionalmutagenesis or vigorous immune reaction, are avoided. SinceVegfa mRNA transfection promoted vascularisation of mouseand human islets following transplantation, this approach repre-sents an attractive strategy to improve islet cell transplantationprotocols.

Conclusion

VEGF-A signalling from beta cells towards endothelial cellsis crucial for endocrine vascular maintenance, while signalsemanating from the endothelium appear critical for pancreasdevelopment, insulin secretion, beta cell proliferation and betacell mass adaptation in response to changes in metabolic de-mand. Tight control of VEGF-A expression is essential forislet vessel maintenance and glucose homeostasis. VEGF-Aloss of function results in islet hypovascularisation and im-paired glucose tolerance, while prolonged VEGF overexpres-sion results in islet hypervascularisation, beta cell death andovert diabetes. Restoration of intra-islet VEGF-A levels afterits overexpression induces a process of beta cell regeneration.In the context of islet transplantation, an increase in the ex-pression of pro-angiogenic factors, such as VEGF-A, or theco-engraftment with pro-angiogenic cells, increases graftrevascularisation and significantly improves islet engraftmentin preclinical studies.Novel therapeutic strategies to restore the endogenous betacell mass through beta cell regeneration or transplantation,

could benefit from (1) further dissection of the mechanismsorchestrating beta cell regeneration upon VEGF-A modula-tion, and (2) clinical translation of experimental pro-angiogenic approaches to improve current islet transplantationprotocols. Taken together, the evidence to date suggests thatthe vasculature may prove a suitable target for the develop-ment of new therapies for diabetes.

Acknowledgements We would like to acknowledge all researchers thatcontributed to the field. We would also like to thank our colleagues andmembers of the Beta Cell Neogenesis laboratory. We apologise to scien-tists whose work could not be highlighted because of space limitations.

Funding The authors acknowledge support by grants from the ResearchFoundation Flanders (FWO), the VUB Research Council, StichtingDiabetes Onderzoek Nederland, the European Union Sixth and SeventhFramework Program, theWetenschappelijk Fonds Willy Gepts (WFWG)of the UZBrussel and the European Foundation for the Study of Diabetes.WS is supported by a postdoctoral grant from Agence Nationale de laRecherche (Laboratoire d’Excellence Revive, Investissement d’Avenir;ANR-10-LABX-73).

Duality of interest The authors declare that there is no duality of interestassociated with this manuscript.

Contribution statement WS and NDL were responsible for drafting thearticle. All authors revised it critically for important intellectual content.All authors approved the version to be published.

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